Molecular and biochemical characterization of rice developed through conventional integration of nDart1-0 transposon gene

Mutations, the genetic variations in genomic sequences, play an important role in molecular biology and biotechnology. During DNA replication or meiosis, one of the mutations is transposons or jumping genes. An indigenous transposon nDart1-0 was successfully introduced into local indica cultivar Basmati-370 from transposon-tagged line viz., GR-7895 (japonica genotype) through conventional breeding technique, successive backcrossing. Plants from segregating populationsshowed variegated phenotypes were tagged as BM-37 mutants. Blast analysis of the sequence data revealed that the GTP-binding protein, located on the BAC clone OJ1781_H11 of chromosome 5, contained an insertion of DNA transposon nDart1-0. The nDart1-0 has “A” at position 254 bp, whereas nDart1 homologs have “G”, which efficiently distinguishes nDart1-0 from its homologs. The histological analysis revealed that the chloroplast of mesophyll cells in BM-37 was disrupted with reduction in size of starch granules and higher number of osmophillic plastoglobuli, which resulted in decreased chlorophyll contents and carotenoids, gas exchange parameters (Pn, g, E, Ci), and reduced expression level of genes associated with chlorophyll biosynthesis, photosynthesis and chloroplast development. Along with the rise of GTP protein, the salicylic acid (SA) and gibberellic acid (GA) and antioxidant contents(SOD) and MDA levels significantly enhanced, while, the cytokinins (CK), ascorbate peroxidase (APX), catalase (CAT), total flavanoid contents (TFC) and total phenolic contents (TPC) significantly reduced in BM-37 mutant plants as compared with WT plants. These results support the notion that GTP-binding proteins influence the process underlying chloroplast formation. Therefore, it is anticipated that to combat biotic or abiotic stress conditions, the nDart1-0 tagged mutant (BM-37) of Basmati-370 would be beneficial.

A monocot semi-aquatic annual grass plant, rice is a member of the Poaceae family and belongs to the genus Oryza 1 . The Oryza genus has 22 wild taxa, of which two species are highly significant for human consumption 2 . These species are Oryza sativa L., generally known as Asian rice, and Oryza glaberrima Steud, popularly known as African rice. Several classifications within the Oryza genus include Oryza officinalis, Oryza ridelyi, and Oryza rufipogon. Of the 22 wild species of Oryza, 15 originated in Asia and 7 in sub-Saharan Africa 3 . These species are beneficial in interspecific rice breeding programs because wild Oryza species have particular traits, including abiotic and biotic stress tolerance 4,5 . Over half of all humans eat rice, making it the most important cereal crop 6 .
Moreover, the full genome sequence of rice has been sequenced with great fidelity, and comparatively high number of transposons were found, making it a great model for the study of transposons 7,8 . An nDart1-0 transposon (pyl-v) was found in a rice virescent rice mutant with light yellow variegated leaves of Taichung-65 (Oryza sativa japonica L.) 9 . In particular, genotypes, containing aDart1-27 an active autonomous DNA element, carry a transposon gene, the element nDart1 are actively transferred throughout the genome 10 . The nDart1 elements, which belong to the superfamily hAT, are excised from chromosomal inserted locations and transposed to other sites, despite having a high degree of sequence similarity, nDart1-0 and the closely comparable non-autonomous

Materials and methods
Plant materials. The required breeding material for the current experiment was collected from two different sources; seed of Indica rice cultivar, Basmati-370 from Rice Research Institute, Kala Shah Kaku, Punjab, Pakistan, and seed of japonica line GR-7895 (consisting nDart1-0 transposon) from Plant Genetic Resources Institute, National Agricultural Research Center (NARC), Islamabad, Pakistan. It has been confirmed that the experimental data collection complied with relevant institutional, national, and international guidelines and legislation with appropriate permissions from Plant Genetic Resources Institute, National Agricultural Research Center (NARC), Islamabad, Pakistan. The data on search experiments revealed with the studies on Basmati-370 have shown that it does not carry any of the aDart elements. The aDart is actually a carrier for nDart1-0 transposon element. For study purposes, mutants were developed for gene tagging by using insertion of the transposon aDart element. A cross was carried out between Basmati-370 and GR-7895 for insertion of aDart element on Basmati-370 from GR-7895 genotype. Dry mature seeds from both lines were surface sterilized for this purpose and raised in controlled environments in pots. To synchronize flowering for crossover, Basmati-370 seeds were cultivated at one-week intervals for four successive weeks. The crossing was carried out by keeping Basmati-370 as a female parent. The F 1 seeds were grown under controlled conditions and backcrossed with designated female plants of Basmati-370 to produce BC 1 F 1 and continue this backcrossing to produce BC 4 F 1 (Supplementary material: Fig. S1). The harvested seeds from BC 4 F 1 were grown and self-pollinated to get BC 4 F 2 . The seeds from BC 4 F 2 were harvested and further grown under controlled conditions to get segregating population upto BC 4 F 4 of plants to identify the abnormal or albino phenotypes used for gene tagging.
Isolation and quantification of DNA from the transposon-induced mutants. The DNA was extracted from the mutant plants through using a protocol of DNA isolation given by Doyle 24 . The extracted DNA was then transferred into eppendorf; along with added 7 μl of RNAase, which incubated at 37 ℃ for 60 min. The quality of DNA was verified using 2% agarose gels, while its quantity was measured using Nanodrop. The samples were then diluted for uniform and standard concentration of DNA up to 1 μl of each sample per 50 µl dH 2 O. The isolated DNA was then quantified by taking a reading of absorbance at 260 nm wavelength keeping using dH 2 O as a blank sample.
RNA extraction and qRT-PCR analysis. RNA was isolated from leaves of rice plants from  (WT) and BM-37 (mutant) through the TRIeasyTM Total RNA Extraction Reagent Kit (Yeasen Biotechnology Co., Ltd. Shanghai, China) as per the manufacturer's guidelines. The quality of RNA was verified using 2% agarose gels, while its quantity was measured using Nanodrop. The cDNA was synthesized by an improved qRT-PCR technique derived from the total mRNA 25,26 . PCR was performed for amplifing of full length cDNA of GTP using specific primers. Oligonucleotide primers (Supplementary material: Table S1) for the amplification of fulllength cDNA of OJ1781_H11 were designed based on the reported sequence in rice (Accession No. AC120986). PCR amplified sequence was cloned and sequenced. The sequence comparison and data analysis were performed using blast with the NCBI database.
We examined the expression level of nDart1-0 in Basmati-370 (BM-37) mutant plants, including plumules, third and fifth leaves at the seedlings stage, roots, stems, sheath, young panicles, and flag leaves at the heading stage of plants. Quantitative  Measurement of levels for chlorophyll pigments, carotenoids and gas exchange parameters. Utilizing a spectrophotometer by making minor adjustments in the methods of Arnon and Wellburn 28,29 , the carotenoid (Car), total chlorophyll, and chlorophyll (a and b) were assessed. In a nutshell, 0.2 g samples from leaves of seedlings grown under controlled circumstances at the three-leaf stage were collected and homogenized for 18 h in the dark in 5 ml of ethanol:water:acetone (4:1:5) solution. Centrifugation was used to get rid of leftover particles. A UV5100 Spectrophotometer was used to evaluate the supernatants. In addition, the portable photosynthetic system LI-6400 was used to quantify the transpiration rate (E), the concentration of intracellular CO 2 (Ci), stomatal conductance (g), and net photosynthetic rate (Pn).
Determination of endogenous phytohormones. The total phenolic contents were calculated using the Folin-Ciocalteu method, with gallic acid acting as a controlled chemical 30 . Equivalents of gallic acid per gram of FW-fresh weight were used to indicate the amount of total phenolic compounds. By employing catechin as a reference ingredient, the aluminum trichloride technique was used to measure the flavonoids concentration 31 .
(gE catechin.100 g1 FW) was used to express the overall flavonoid content. Following the manufacturer's recommendations, we used a plant ELISA kit to detect the endogenous salicylic acid (SA), abscisic acid (ABA), gibberellic acid (GA) and cytokinins (CK). To measure the concentrations of SA, ABA, GA and CK in the samples, leaves were pulverized in liquid nitrogen. The hormonal SA, ABA, GA and CK ELISA kits come with a set of calibration standards. The SA, ABA, GA and CK levels in the samples were then measured by fitting the standard curve and generating a comparable trend in the samples.

Measurement of antioxidants, H 2 O 2 and malondialdehyde (MDA) contents. The leaf samples
were homogenized in sodium phosphate buffer (pH 7.8), centrifuged at 13,000 rpm for 20 min at 4 °C. Then superoxide dismutase (SOD) activity was measured spectrophotometrically as described by Zhang et al. 32 , at 560 nm by assessing the ability of each unit to inhibit 50% photochemical reduction of nitro blue tetrazolium chloride (NBT). Peroxidase (POD) activity was determined according to Zhou and Leul 33 , at 470 nm and the changes related to guaiacol were normalized with the activity constant (ε = 26.6 mm) and catalase (CAT) activity was determined according to Aebi 34 . The APX activity was calculated depending on the decrease in absorbance at 290 nm according to Nakano and Asada 35 as ascorbate was oxidized. This study measured reactive oxygen species in plant tissues, including hydrogen peroxide (H 2 O 2 ). Hydrogen peroxide content was measured as described by Velikova et al. 36 . Malondialdehyde (MDA) content was determined according to Morales and Munné-Bosch, 37 . The total phenolic content was measured by the Folin Ciocalteu method using gallic acid as a reference compound 30 . The flavonoids content was determined by the aluminum trichloride method using catechin as reference compound 38 .
Statistical analysis. The standard error and mean from three replications are reported for each set of data.
Data analysis was carried out using Statistics 8.1, a statistical software programme. The recorded data was analyzed through one way variance analysis and LSD test and Tukey's test to determine pairwise significance at p ˂ 0.05. Origin Pro (version 8.5) was used to create the graphs.

Results
To study gene action, the regulation and characterization of genes have become the most important genetic tool in biotechnology and genetic engineering due to reverse and forward genetic approaches. For the evaluation of genetic potential and variations in the population of crop plants, there is necessary to develop mutants by using irradiations, chemicals, transposons, and induced genetic mutations for tagging genes to carry out functional genomic evaluations of species genomes from narrow to more comprehensive ranges. In our current study, we have described a active, and non-autonomous genomic or DNA transposable element in rice, named nonautonomous DNA-based active rice transposon one (nDart1). The nDart1 is a causative transposable element of allele with spontaneous mutable and virescent nature, the pale-yellow-leaf variegated or pyl-v, which caused pale www.nature.com/scientificreports/ yellow with dark-green streaks on leaves of rice plants at very early seedling stages usually due to the expression of nDart1 gene in rice. There are multiple transposon elements along with their multiple gene insertional sites, which a crop plant line of genotype has been found in limited numbers because of the reduced requirements for the identification and characterization of specific genes tagging a population genome for achieving maximum saturation of transposable elements. For our current study, we have selected the nDart1 gene to produce rice mutants, which were expressed as pale-yellow-leaf variegated. The nDart1 gene was inserted into the Basmit-370 variety through the conventional backcross breeding method.
Development and phenotypic appearance of mutants. In our current research study, we have utilized an endogenous and available transposon named nDart1 to develop mutants for tagging genes in the Basmati-370 rice genotype. As we could not find any active nDarts or active autonomous element Darts in local basmati varieties, we introduced japonica-derived aDart and nDart1-0 into the indica rice variety Basmati-370 through backcrossing for mutant genes. Different plants were selected from segregating populations that showed variation and were tagged as mutants (Fig. 1A) from the BC 4 F 4 population. Through genetic analysis, this mutant was surmised to be controlled by a two-component transposon system. During growing of rice plants in the field after BC 4 F 4, there were normal green leaves in the WT and variegated types of albino rice plants in BM-37 (Fig. 1B, C), which was introduced with nDart1/aDart1. The lines which showed gene expression were postulated to have a recessive allele that had the nDart1-0 homozygous as well as an active autonomous element as aDart1 with heterozygous allele conditions. It was found that the mutated line produced stable albino progenies (Fig. 1D). The stable mutants indicated that there were not only stable GTP homozygous alleles but also having without an aDart1element with a ratio of 3:1 for F 2 . It was also found that there was no difference between the WT as well as the mutants in rice seeds, but the difference was found only at the stage of seedling growth, where the normal seeds produced normal green leaves and stem, whereas the mutated seeds produced white shoots and leaves. The results indicated the presence of aDart1 elements as found stable in rice plant phenotypes. It was found that aDart1-dependent nDart1-0 somatic excisions of allele produced a leaf with variegations that developed throughout cell lineage or progenies of mutated individuals of rice plant. However, the white mutated or variegated plants may grow their panicles with fertile grains under extreme conditions. Gene tagging by nDart1-0-iPCR method. The frequency of spontaneous mutation under natural growth conditions is extremely low. Transposons are useful for gene tagging and functional analysis to create natural variation, so it is useful to explore active transposon sources. Mutant phenotypes were tagged in the field and DNA was isolated from phenotypically variegated leaves of mutants. DNA was subjected to restriction digestions and self-ligation. Data search revealed that nDart1-0 has thirteen homologs and a high homology above 99%. At position 254 bp, nDart1-0 has "A", whereas the homologs have "G" as shown in Table S3 (Supplementary material). The site "A" was used efficiently to distinguish nDart1-0 from its homologs.
Keeping in view that according to this technique, DNA is restricted with Alu I, self-ligated, and again subject to another restriction digestion with Bmt I and amplification by iPCR using specific primers. Digestion by Alu I, restrict nDart1-0 at position 254 as it carries a unique specific restriction site due to the substitution of a base pair "A" whereas homologs carry "G" at this site during self-ligation, this digested fragment of the nDart1-0 ligates with a fragment of the gene invaded by him (Fig. 2). Self-ligation yields some specific fragments having a part of the nDart1-0 and the gene invaded by him along with other fragments. Again, restriction of this self-ligated DNA by Bmt I cleaves the nDart1-0 as it carries Bmt I site, as a result, we get some fragment of the invaded gene flanked by nDart1-0. These fragments were amplified through iPCR using the specific primers designed for flanking region of nDart1-0. The two rounds or cycles of iPCR (like pre-selective and selective gene amplifications) were carried out for confirmation of results. During the selective type of amplification, the www.nature.com/scientificreports/ further enhancement and enrichment of transposon elements or their related specific fragments were achieved through using available PCR master mix products like a template to carry out the second transposable element amplification with the nested type of primer, which specifically recognized highly conserved transposable element regions in the genome. Nonspecific genomic rearrangements during iPCR are ruled out because of the primers derived from specific regions of the nDart1-0. On the other hand, the nDart homologues does not carry Alu I site at position 254, they are not digested at this position and consequently are not amplified with specific primers during iPCR (Supplementary material: Fig. S3A). When DNA restricted with Alu I and self-ligated is used as template for iPCR, nDart1-0 and all its homologues are amplified but this amplification is restricted to nDart1-0 if self-ligated DNA is again restricted with Bmt I. For confirmation of fragments obtained either these were transposable elements or not detected transpose element fragments were amplified to study the transposon element flanking sequences, PCR amplified fragments were sequenced and analyzed. The amplification patterns for each fragment were similar i.e., flanked by nDart1-0, except for the difference in genes invaded. So far, we have detected 12 genic regions invaded by nDart1-0 in BM-37 mutant (Supplementary material: Table S3). It was found that there was a mutant amplified fragment which was containing nDart1-0 flanking sequences were detected which were derived due to the insertion of nDart1-0 on to the first exon regions of a putative gene (Supplementary material: Fig. S3B).
The results from our study have suggested that the nDart1-0 element insertion as well as its excision into the putative gene was as OJ1781_H11 on chromosome 5. Blast analysis of the sequence data revealed that GTPbinding protein locus on the BAC clone OJ1781_H11 of chromosome 5 was found to contain an insertion of DNA transposon nDart1-0. This gene has a nucleotide sequence of 2701 bp and 918 cDNA. The ORF encoded a polypeptide of 305 amino acid sequence and comprises 7 exons interrupted by 6 introns. During the process of gene tagging five revertants were found. These revertant phenotypes were analyzed to find out absence or presence of the transposon. DNA was isolated from the revertant phenotypes and transposon-inserted regions were amplified by the already-designed primers from the flanking regions of the transposon. Amplified fragments (Fig. 3A, B) were cloned and sequenced. Sequence analysis revealed that the transposon moved from the insertion point, leaving behind the footprints.

Expression analysis of BM-37 mutant.
To study the expression pattern of transposon nDart1-0, the qRT-PCR was carried out on several tissue levels (3rd and 5th leaves at the seedling stage, plumules, sheath, shoots, roots, young panicles and flag leaves at heading stage)of BM-37 mutant. The highest expression was found in the third leaves stage of seedling, compared to the fifth and flag leaves, across all the tissues analyzed, indicating that early seedling leaves had higher expression than other tissues. The tissues, including stem, sheath, and roots, expressed significantly lower expression level than leaves, while young panicles and plumules showed www.nature.com/scientificreports/ a minor level of expression for BM-37 (Fig. 4). The findings suggested that nDart1-0 have an influential role in leaf chloroplast development particularly during the early seedling stage.

Ultrastructural analysis of mesophyll cells. Using TEM, the upper part of leaves at the tillering stage
were studied to examine the ultra-structural alterations in the chloroplast of WT and BM-37 mutant plants. In contrast to the BM-37 chloroplasts, which showed well-developed lamella structures, the grana lamella stacks in WT plants were sparse and sparsely populated. This suggests that the BM-37 mutation had a significant impact on biogenesis. The outcome also demonstrated that starch granules were difficult to locate in the chloroplast of the BM-37 mutant (Fig. 5A, B), although they were present normally in the WT. In the BM-37 mutant, there were more osmophilic plastoglobuli (OP) than in the WT, indicating that the chlorophyll metabolism was substantially compromised.
Transposon n-Dart1-0 gene interrupted the pigment metabolism and photosynthetic efficiency. At the seedling and heading phases of the WT and BM-37 plants, the carotenoid and chlorophyll contents were examined to ascertain the impact of the nDart1-0 transposon on photosynthesis pigment metabolism. The chlorophyll (a, b and a + b) contents and carotenoid levels were drastically decreased in BM-37 at seedling stage as compared with WT; however, the chlorophyll contents and carotenoids showed non-significant results at the heading stage in both WT and BM-37 mutant plants (Fig. 6A, B). Additionally, the gas exchange parameters were measured to compare the photosynthetic characteristics of WT and BM-37 mutant plants. As compared to WT, the BM-37 mutant dramatically lowered the rates of photosynthetic activity (Pn), stomatal conductance (g), intercellular CO 2 concentration (Ci), and transpiration (E) (Fig. 6C-F).    (Fig. 7A, B). We also studied the expression level of different genes related to chloroplast development, including RpoA, RpoB, OsRpoTp, Rps7, Rps20, RNRL, RNRS, OsPolP1, FtsZ and Rpl21 [41][42][43][44][45][46] . Relative expression levels of all genes associated with chloroplast development were significantly down regulated in the BM-37 mutant as comparable to WT plants (Fig. 7C). Under abiotic stress, it's possible that these important genes' aberrant expression caused the mutant phenotype. The findings showed that the BM-37 mutation had a significant impact on the metabolism of chlorophyll, photosynthesis and chloroplasts development in BM-37 mutant cells.

Ultra-structural alterations in chloroplast caused oxidative damage. The BM-37 mutant and WT
leaves at seedling stage were used to assess the levels of the most prevalent growth-related phytohormones, such as SA, CK, GA and ABA in the plant. According to the findings, CK levels were substantially lower and GA and SA contents were significantly higher in the BM-37 mutant than in the WT, while ABA levels did not differ significantly from those of the WT (Fig. 8A-D). According to the findings, the BM-37 mutant displayed efficient hormonal signaling for chloroplast formation.
We also evaluated ROS, such as H 2 O 2 production and MDA contents, and their scavenger antioxidants, such as lower molecular weight antioxidants and enzymatic antioxidants, in order to study the activities of antioxidants for comparison between WT and BM-37 mutant (Fig. 8E-L). Enzymatic antioxidants like CAT and APX, which are significantly lower in leaves of BM-37 while SOD is considerably higher in leaves of the WT, might scavenge the oxidative compounds (Fig. 8E, G, K). MDA levels were substantially enhanced in BM-37 plants than in the WT plants (Fig. 8L), showing that mutant leaves had more oxidative damage. The results also showed that there are no appreciable changes between BM-37 and WT values for H 2 O 2 and POD (Fig. 8I, J). The results also depicted that the values of TFC and TPC also significantly reduced in BM-37 mutant leaves as compared with WT leaves (Fig. 8F, H). The study found that BM-37 had inadequate antioxidant defense because it was less effective than the WT to scavenge ROS generated by defective chloroplasts.

Discussion
For gene-tagging, insertional mutagenesis by T-DNA, Tos17 or maize transposons, Ac/Ds and En/dSpm is very useful, but main disadvantage is the frequent presence of somaclonal variation. The frequency of gene tagging by endogenous retrotransposon Tos17 is very low 47,48 . Mutations by inserting endogenous DNA TEs are desirable to produce transposon-tagged mutant resources. Only the disadvantage with an endogenous DNA TE is to generate stable mutants induced by footprints. In this case, it is difficult to identify the mutant gene. Previously efforts were made to reactivate mPing, a tourist-like thhe tourist-like MITE family member, in the rice genome through tissue culture techniques 49,50 and gamma-ray irradiations [51][52][53] . Insertion of mPing induced duplications of targeted site (TSDs) of TTA or TAA, and mPing usually tend to insert into the non-coding region or intron, so mPing was not considered a good transposon-tagging tool. On the other hand, DNA TEs, nDat1-0 were discovered in rice 9 . Structurally, nDart1-0 is non-autonomous active DNA transposon with a small size (607-bp), and highly rich GC (about 72%), and tends to transpose into gene regions, it carries a 19-bp perfect segment of terminal inverted repeats (TIRs), which usually has 8-bp high GC targeted site duplications and belongs to hAT super family 54,55 . The nDart1-0 inserted in OsClpP5 was also found to invade two other genes, indicating its high efficiency for gene-tagging 9 . The blast search revealed that Nipponbare and indica variety 9311 carries 13 and 7 nDart1 homologs, respectively. The efficient nDart1-0 utilization for gene-tagging usually depends upon aDart www.nature.com/scientificreports/ presence or occurrence in that rice variety. It was observed that Nipponbare carries 63 candidates of autonomous element aDart 56 . These specific features distinguish nDart1-0 from most of the last and previously studies and discoveries of non-autonomous transposable elements in DNA. The transposable elements caused variegation or albino plants, when these elements expressed in rice varieties 57 . In maize and Antirrhinum majus, the transposon tagging genes have been reported, which have been successfully isolated along with different ranges of transposable genes or elements [58][59][60] . Therefore, we developed a strategy to tag genes mutated by the invasion of nDart1-0. The newly developed strategy using iPCR (n1-0SPiPCR) is useful for our nDart1-0-specific tagging system. By this system, the transposons elements are usually tagged through an inverse PCR by restriction-ligation-restriction-mediated, which starts from specific sequences of transposon, and amplifies the part of flanking sequences to a specific site for restriction. The resulting PCR products may be used to clone, analyze or sequenced [61][62][63] . The previous studies elaborated that both aDart and nDart1-0 have been found useful tool for gene-tagging in japonica and indica rice species. We have found that the utilization of a well-established systematic and computational approach for isolation of active transposon element nDart1-0 from the host cell genome, may help to understand the pattern of inheritance of transposable elements in rice 64,65 . However, this technique not only the fast method for detecting the nDart1-0, while it also has its utilization in gene tagging for various genes in rice genome. www.nature.com/scientificreports/ The albino or variegated mutant plants provide important and valuable information regarding plastid growth and development; their genetic analysis has been difficult because of their higher lethal phenotypic effects 66 . From the present study, the muted allele was successfully identified by utilizing the recently developed procedure named as nDart1-0-iPCR. It has been found from tagging for nDart1/aDart1 line that it contained the most frequent and repeated transposable element nDart1-0 while the other line had nDart1 transposable elements 67,68 . It has been found that nDart1 transposable elements showed their behavior to integrate within the promoter regions of genes 69 . Insertion of nDart1-0 also was found to be a cause for mutation in GTP gene at 13 bp downstream from the initiation ATG codon of GTP gene. There was a frameshift mutation in GTP gene due to nDart1-0 insertion downstream of GTP gene transcriptional initiation sites. The results from previous research experiments have also revealed that the nDart1 insertion at the transcriptional initiation sites caused effects on the expression levels in downstream gene sites. The OsClp5 has shown its effects which were disrupted due to insertion on 5'-UTR position of nDart1 transposon in pyl-v mutant 70 . It was found that mutant rice line exhibited an increased and enhanced inflorescence, indicating that the nDart1-0 insertion may cause up-regulated level of gene expression from downstream sites of gene names such as Aberrant Panicle Organization1 in rice [71][72][73] .
From various research studies, the GTP protein is required in higher amounts for the biogenesis of chloroplast while lower amount for etioplast biogenesis. Translation efficiency of GTP has been found to be activated from etioplast during chloroplast development. The cell division of plastids mostly occurred during P0 up to the very early P4 growth stages, while the activation for photosynthetic apparatus has been found during P4 growth stage of rice liens [74][75][76] . Our current study suggests that the GTP protein functions during the development of thylakoid membranes of chloroplasts due to a well-established genetic system of plastids. While in the non-green plant tissues, it has also been found that the GTP protein abundance and accumulation during chloroplast development was regulated at the post-transcriptional level.
The BM-37 mutant, with insertion of nDart1-0 have lower contents of carotenoid and chlorophyll at seedling stage and a major reduction in pigment would probably affect the chloroplast development and ultimately www.nature.com/scientificreports/ photosynthesis process (Fig. 6A). Transmission electron microscopy confirmed ultrastructural changes in BM-37 due to mutation of nDart1-0 gene (Fig. 5). More number of plastoglobuli observed in BM-37 that is consistent with the previous research that plastoglobuli number increased when mutants have defect in thylakoid membrane biogenesis and their formation prevent thylakoid from oxidative damage 77 . In BM-37, the chloroplast contained smaller starch granule, indicating that starch formation was disturbed, which affect the energy supply for chloroplast development 78 . Moreover, the reduction in chlorophyll contents of BM-37 mutant have decreased the key photosynthetic parameters, such as the net photosynthetic rate, stomatal conductance, transpiration rate and intercellular CO 2 concentration (Fig. 6C-F) which was also observed in mutant where gas exchange parameters (Pn, Tr, g and Ci) decreased with reduction in chlorophyll contents 79 . In addition, lower transcript level of genes involved in chloroplast development, chlorophyll biosynthesis and photosynthesis of BM-37 mutant ( Fig. 7A-C) have provided the evidence that mutation in nDart1-0 has disturbed plastid-to-nucleus retrograde signaling which is consistent with the previous finding that chloroplast retrograde signaling was also disrupted in spc1 mutant of Arabidopsis 80 . Taken together, these results indicated that mutation in BM-37 was associated with chloroplast development and photosynthetic efficiency. The BM-37 mutant plants at seedling stage lead to substantial reduction of chlorophyll and carotenoids contents resulted in impaired chloroplast development that could accumulate higher ROS. Carotenoids provide protection against photo oxidative damage by detoxification of excessive free radicals and reactive oxygen species (ROS) 81 . For instance, impaired chloroplast biogenesis in leaf variegated rice zebra2 25 as well as mutation in ACD2 encoding red chlorophyll catabolite reductase 82 resulted in excessive accumulation of ROS. Plant produce anti oxidative enzymes to detoxify ROS such as SOD to cope with superoxides while CAT, POD and APX to scavenge H 2 O 2 . Moreover, it has been reported that non-enzymatic antioxidants can be oxidized to monodehydroascorbate under higher ROS level 83 . Accordingly, our results showed lower contents of non-enzymatic antioxidants (Fig. 8F, H) in BM-37 mutant plants. Hence, the results inferred that malfunctioning of ROS scavenging enzymes and accumulation of H 2 O 2 and MDA due to impaired chloroplast development has introduced the yellow leaf color in the BM-37 mutant at seedling stage.
Under various abiotic stresses, the reactive oxygen species (ROS) and lipid peroxidation produced in crop plants which caused damages in cells, and lead to the death of crop plants. The reduction in growth results from cellular and subcellular abnormalities in plant cells [84][85][86] . Each crop plant cell has the ability to produce antioxidants (SOD, POD, CAT and APX) to induce the self-defense mechanism in cell, especially for chloroplast and mitochondria 87,88 . The reactive oxygen species (H 2 O 2 ) mostly affects the membranes of chloroplast and mitochondria, which ultimately caused death of cell. The production of peroxidase (POD), catalase (CAT), guaiacol peroxidase (GPOD), hydrogen peroxide (H 2 O 2 ), superoxide dismutase (SOD), lipoxygenase (LOX), catalase (CAT), ascorbate peroxidase (APOD) and glutathion-S-transferase (GST), glutathione, proline, ascorbic acid, etc. are bioorganic compounds produced in cells 89,90 . The GTP proteins provide an environment for these antioxidants to work under various conditions. The GTP proteins are localized in the cells where there is a need to combat stress conditions. Exploration of various studies on plant hormonal regulation and G-protein signaling reveals several intriguing similarities in their functional roles. In addition, Arabidopsis G-protein mutants display altered www.nature.com/scientificreports/ phenotypes in response to different plant hormones 91,92 , and transcriptome data reveals a significant change in the expression of G-protein genes under different hormonal treatments 93 .From these studies, it is concluded that transposon nDart1-0 is a useful system to develop mutants for functional gene analysis, and n1-0 SPI PCR is a powerful tool to analyze mutants developed by the insertion of transposon nDart1-0. The transcripts of GTP binding protein were observed in various tissues of plants, usually in the early stages of plants. Era-like GTP binding protein is a new member of an old family, and it is vital for chloroplast biosynthesis and plant survival.

Conclusions
These studies conclude that transposon nDart1-0 is a useful system for developinga useful system for developing mutants for gene functional analysis and n1-0 SPI PCR is a powerful tool to analyze mutants developed by the insertion of transposon nDart1-0. The transcripts of GTP binding protein were observed in various plant tissues, usually in the early stages of plants. Era-like GTP binding protein is a new member of an old family, and it vital for chloroplast biosynthesis and plant survival. The protocol described may be helpful as a potential method for identifying transposable elements from other organisms, including plants like maize. However, it might have been hoped that this technique described not only the fast method for detecting the nDart1-0 but also its utilization in gene tagging for various genes in the rice genome.

Data availability
All of the data generated or analyzed has been provided in the manuscript and supplementary files.